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Abstract

For the first time, through validation of the modified Chromatin
Immunoprecipitation (ChIP) method (in vitro ChIP), the direct binding of NPR1 to
the PR1 promoter was demonstrated. This is a novel advancement on plant systemic
acquired resistance (SAR)-mediated disease responses. The NPR1 protein (nonexpressor
of pathogenesis related gene 1) is a transcriptional co-activator and positive
regulator of SAR, a long-lasting mobile defense signal found in plants. The
pathogenesis-Related gene 1 (PR1) is particularly induced during defense response,
and as such, is typically used as a marker for establishment of SAR in plants. Salicylic
acid (SA) is a phytohormone required for SAR-mediated defense responses against
pathogens. Recently, the role of NPR1 as a SA receptor was demonstrated; SA has
been shown to directly bind to NPR1 through Cysteine 521 and 529 on the Cterminus
region of NPR1 via the transition metal copper. The binding of SA to NPR1
results in disruption of the interaction between BTB/POZ and the C-terminus domains
of NPR1. Upon SA-NPR1 binding, the C-terminus transactivation domain is released
from the auto-inhibitory BTB/POZ domain, resulting in activation of the NPR1
transcription co-activator function, followed by PR1 transcription in Arabidopsis
thaliana. Arabidopsis thaliana has an inducible defense system and is considered a
model plant for studying disease resistance responses.
In the current research, NPR1 was demonstrated to bind to the PR1 promoter
at two distinct regions, in the presence and absence of SA. In the presence of SA, the
binding site of NPR1 was determined to be localized at the -636 to -646 base pair
sequence; however, in the absence of SA, NPR1 was found to bind around the -790 to
-833 base pair sequence. In addition, two distinct DNA binding domains were
identified within NPR1, localized on the C and N-terminus regions. In the absence of SA, the DNA binding domain within the N-terminus region, located between amino
acids 110-190, was shown to facilitate the binding of NPR1 to the PR1 promoter
through the amino acid cysteine 150 (Cys150) via transition metal. The DNA binding
domain on the C-terminus region, located between amino acids 513-535, was
demonstrated to allow the binding of NPR1 to the promoter of PR1 in the presence of
SA. Two amino acids, cysteine 521 and 529 (Cys521/529), were shown to be essential
for SA binding to NPR1 and subsequent NPR1 binding to the PR1 promoter.
Furthermore, 4hydroxy benzoic acid (4-OH-BA), the inactive analogue of SA, has
been demonstrated to be a potent inhibitor of NPR1-PR1 promoter interaction, both in
vivo and in vitro, by competing with SA for NPR1 binding. Moreover, we
demonstrated that other analogues of the NPR1 protein, NPR2, NPR3, and NPR4, are
also recruited to the PR1 promoter. NPR4 showed a similar binding profile to NPR1,
both in the presence and absence of SA. NPR2 and NPR3 were observed to only
interact with the PR1 promoter in the absence of SA. Both NPR5 and NPR6 were
shown to forgo binding to the PR1 promoter, further confirming their role in plant
developmental processes other than defense. In addition, the binding of NPR1 to the
PR1 promoter was demonstrated to be conserved among other plant species, including
rice and maize. Both rice and maize NPR1 proteins were observed to bind to the PR1
promoter in the presence of SA and a metal co-factor, similar to Arabidopsis NPR1.
Our results expand our understanding of how NPR1 interacts with the PR1
promoter to regulate gene expression during SAR establishment. This study also
revealed that NPR1-mediated SAR defense signaling is conserved among other crop
species, which can potentially facilitate the identification of novel plant-priming
compounds through high-throughput chemical screening procedures alongside the
application of the validated in vitro ChIP technique as a primary screening method.